GSM7118728: E15-Col10a1; Mus musculus; RNA-Seq
Bones were isolated from limbs and minced with scissors or tweezers. The fragmented bones were incubated in DMEM containing Collagenase P, Liberase TH and DNase I at 37ºC with gentle shaking for 1h. Dissociated cells were collected every 20 min and strained with a 100 μm nylon mesh. Cells were collected by centrifuge and resuspended in FACS buffer (2% FBS/2 mM EDTA/PBS). For experiments on bone marrow cells prepared from 3-week-old mice, red blood cells were removed by 0.165 M ammonium chloride treatment. The isolated cells were stained with antibodies against CD45, Ter119 and CD31. Fixable viability dye (eBioscience) was used to stain dead cells. Cells were sorted for single cell 3' gene expression library construction. Library construction was performed according to the manufacturer's instructions (single cell 3' v3.1 protocol, 10x Genomics) with slight modification. Cells up to 10,000 were directly sorted into the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM).